Robust expression of multi-subunit protein complexes is crucial for understanding the basic biology of organisms. Expression of the individual components in E. coli or yeast may be quick and cost-efficient, but many mammalian proteins show poor expression or solubility when expressed in these hosts. Further, they lack appropriate post-translational modifications, which may be essential for proper activity or interaction with other proteins. Current mammalian expression systems typically express one protein per plasmid, maintained either in the cytoplasm or randomly integrated onto the chromosome. Stable and coordinated expression of these proteins is not possible. Further complications arise in monitoring and analyzing complexes formed from the recombinant proteins. The goal of this Phase I proposal is therefore to develop a system to conditionally express multiple proteins as a mammalian operon on a stable mini chromosome. The expression vector will be based on the linear pJAZZ vector, which removes functional and structural barriers to cloning. In addition, we will develop novel bacterial phytochromes (PHYs) as red and near-infrared fluorophores to monitor and quantify transient interactions among proteins. These PHYs will be useful for fluorescent imaging in vivo and in denaturing gels. Moreover, they will have FRET capability. This system will enable a wide range of studies on structure, function, assembly, and interaction of proteins in multi-subunit complexes. It will also facilitate identification of binding partners and improved imaging of cellular structures. PUBLIC HEALTH RELEVANCE: A large number of proteins within cells form unique multi-subunit complexes, which are typically very difficult to study. We propose to develop a novel system to readily produce and monitor protein complexes in cell cultures. This work will enable researchers to better understand protein interactions in normal and disease situations.